PROJECT SUMMARY Chromatin-based DNA damage response (DDR) mechanisms are fundamental for preventing genome and epigenome instability, which are hallmarks of cancer. How chromatin promotes genome-epigenome integrity in response to DNA damage is a critical question. This proposal aims to address this question by comprehensively analyzing the involvement of all 42 human bromodomain (BRD) proteins in the DDR. The BRD is the primary reader domain of acetylation. Chromatin acetylation is a key signaling event involved in detecting, signaling and repairing DNA damage. Thus, BRD proteins represent attractive candidates for reading damaged chromatin to mediate genome-epigenome integrity. Our complete analysis of BRD protein dynamics at DNA damage sites provides an unprecedented view of the involvement of BRD proteins in the DDR. From our studies, we identified one-third of BRD proteins relocalized upon DNA damage, a phenomenon common to DNA damage factors. These findings demonstrate the widespread involvement of BRD proteins in the DDR and provide an experimental framework to further identify the function of BRD proteins in the DDR. We identified the BRD protein ZMYND8 in a novel transcription-dependent DNA damage recognition pathway. ZMYND8 recognizes and represses actively transcribing damaged chromatin by recruiting NuRD chromatin-remodeling complexes to these sites to facilitate repair by homologous recombination. Additional studies based on our results are poised to provide critical mechanistic insights into how damaged chromatin is recognized and processed to promote DNA repair. We will then build upon our identification of other DNA damage recruited BRD proteins, including TRIM24-TRIM28-TRIM33, to identify additional DDR pathways involving BRD proteins. Several BRD proteins are linked with cancer, suggesting these studies will not only provide insights into their DDR functions but will also provide vital information for their involvement in cancer. We will also test our hypothesis that non-DNA damage recruited BRD proteins are involved in the DDR. These studies will provide an unprecedented understanding of how BRD chromatin reader proteins orchestrate chromatin-based DDR pathways to protect the integrity of the genome. This work could impact cancer biology as ZMYND8 and NuRD are often mutated in cancer and histone acetylations bound by ZMYND8 are deregulated in several cancers. BRD proteins and DDR factors are both actively being pursued as therapeutic targets. The pre-clinical success of small molecule inhibitors targeting the BRD (e.g. BRD4 inhibitors JQ1 and BETI) has made drugging the epigenome by targeting BRD proteins a major direction for drug discovery. Our identification of BRD proteins in the DDR has important implications for targeting these proteins in cancer. Information gained from this proposal will help guide therapeutic strategies for targeting BRD proteins as transcriptional regulators and mediators of the DDR in cancer.